Spermatozoa throughout the animal kingdom are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid within an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Little is known about how individualization is carried out. However, the importance of one aspect of this process for human fertility, the elimination of excess cytoplasm, is suggested by the fact that many conditions or treatments resulting in infertility disrupt this process. We recently reported that multiple caspase family proteases and their activators were required for spermatid individualization in Drosophila. These observations were striking because caspases are the core of the evolutionarily conserved, apoptotic cell death machine. Once activated they typically cleave a number of cellular substrates that ultimately lead to cell death and corpse phagocytosis. Our observations raise a number of questions, in particular, 1) How is it that spermatids avoid death in the presence of active caspases that would induce apoptosis in other cells? 2) What are the pathways that mediate caspase activation in spermatids? We have proposed three specific aims to address these questions. We will: 1) characterize the mechanisms that spermatids utilize to avoid apoptosis in the presence of activated caspase; 2) identify the mechanisms that mediate caspase activation in spermatids; and finally, 3) we will characterize mutations derived from a recent, large scale screen for male sterile flies, with the goal of identifying new regulators of caspase activity and function in spermatogenesis. Where studied, signaling pathways in flies and mammals utilize similar components and regulatory mechanisms. Therefore, it is likely that successful completion of the proposed experiments will increase our understanding of how spermatogenesis is regulated in human health and disease. In addition this work is likely to provide insight into the mechanisms by which caspase activity, and the consequences of caspase activation, are regulated.